1

This is an author version of the contribution published on:

Questa è la versione dell’autore dell’opera:

[Field Crops Research, Volume 179, 1 August 2015, Pages 95–102; DOI:

10.1016/j.fcr.2015.04.014.

The definitive version is available at:

La versione definitiva è disponibile alla URL:

[http://www.sciencedirect.com/science/article/pii/S0378429015001380]

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FIELD CROPS RESEARCH

Title: Evaluation of common and durumwheat rheological

quality through Mixolab® analysis after field damage by

cerealbugs.

Authors:

Massimo Blandinoa*, Federico Marinaccioa, Barbara Letizia Ingegnoa, Marco

Giuseppe Pansaa, Patrizia Vaccinob, Luciana Tavellaa, Amedeo Reyneria

Affiliation:

a University of Torino, Dipartimento di Scienze Agrarie, Forestali e Alimentari, Largo

P. Braccini 2, I-10095 Grugliasco (TO), Italy.

b Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Unità di

ricerca per la Selezione dei Cereali e la Valorizzazione delle varietà vegetali (CRA-

SCV), via Forlani 3, 26866, S. Angelo Lodigiano (LO), Italy.

*Corresponding author: Massimo Blandino

Phone +39 011 6708895, massimo.blandino@unito.it

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Abstract

The pre-harvest damage of wheat by sunn pests decreases flour quality. Mixolab® is

a recent instrument that can be used to accurately describe the technological

behaviour of flour dough, since it is able to evaluate both protein and starch

components at the same time. Two-year field experiments were carried out to study

the effect of Eurygaster maura (Heteroptera: Scutelleridae) feeding on the quality

traits of common and durum wheat, recorded using different protocols, in comparison

to the traditional sodium dodecyl sulphate sedimentation test (SSV). In 2012-13

growing season, a damage rate between 16% and 21% of damaged kernels led to a

greater reduction in dough stability for common (-65%) than for durum wheat (-32%),

while the protein strength was affected more by insect activity in durum wheat (-

56%). In 2013-14 growing season, E. maura feeding (on average 32% of damaged

kernels) reduced SSV by 5% and 66%, dough stability by 12% and 30%, and protein

strength by 12% and 16%, for common and durum wheat, respectively. The

increasing percentages of damaged kernels in both crops led to a clear decrease in

dough stability and protein strength; a significant change in the rheological

parameters was noticeable at a 2.5% level of damaged kernels. SSV resulted to be

significantly correlated to Mixolab® dough stability, the protein strength, the protein

network wakening rate (α) and Change in Mixolab Consistency (CMC, “Wheatbug”

protocol) for both common and durum wheat. Therefore, Mixolab® can be considered

a suitable tool for a quick detection of damage caused by sunn pests in common and

durum wheat flour.

Key words

Common wheat; durum wheat; proteins; gluten; Eurygaster maura

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Abbreviations: Change in Mixolab Consistency, CMC; Dough Development Time,

DDT; Grain Protein Content, GPC; GS, growth stage; Sodium dodecyl sulphate

sedimentation volume, SSV; water absorption, WA.

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1. Introduction

Grain protein content (GPC) and gluten quality are key traits that influence the end-

use of wheat market classes throughout the world (Foca et al., 2007). High levels of

proteins as well as gluten quantity and strength are the predominant factors

associated with superior bread- and pasta-making quality. Therefore, these traits are

desirable for the marketability of both common and durum wheat in several supply

chains (Brown and Petrie, 2006; Kovacs et al., 1997). Although the environmental

conditions and the genotype, i.e. the choice of variety, are fundamental for the

technological behaviour of the derived dough, the agricultural practices, and in

particular nitrogen nutrition, also influence the flour quality to a greater extent.

Considerable modifications of wheat quality can also be produced by several species

of insects, in particular cereal bugs, which are also known as sunn pests, belonging

to Scutelleridae (shield-backed bugs) and Pentatomidae (stink bugs) (Critchley,

1998). Eurygaster maura (L.) (Heteroptera: Scutelleridae) is the most noxious

species in western Europe, in terms of reduction in grain quality (Pansa et al., 2015;

Vaccino et al., 2006). Sunn pests feed on wheat during the different stages of

developing grains; in case of early attacks, the damage mainly concerns losses in

kernel weight and consequently in grain yield; late attacks during the grain filling

period, which are more frequent, lead to a reduction in technological quality. In this

case, the insects suck the milky nutrients from the immature grain by piercing it and

injecting proteolytic enzymes, via their saliva, which persist in the flour after milling

and cause the breakdown of the gluten structure in the dough (Olanca et al., 2009).

Thus, the rheological properties of the dough obtained from bug-damaged wheat are

characterized by lower farinographic development time and stability, lower

6

alveographic strength, tenacity and extensibility, and a lower gluten index (Karababa

and Ozan, 1998).

In the case of common wheat, the result is the production of bread with poor volume

and texture (Aja et al., 2004; Pérez et al., 2005), while in the case of durum wheat,

bug damage can negatively affect the cooking potential, especially in varieties with

poor gluten quality (Petrova, 2002). Ozderen et al. (2008) have demonstrated that

the semolina properties and spaghetti quality of durum flour obtained from bug-

damaged wheat decreased significantly as the damage levels increased. Although

there are several reports on the effects of bug damage on common wheat in the

literature, limited information is available related to its effects on durum wheat, and in

particular a direct field comparison of the bug feeding effect on both crops is missing.

The relevant reduction in technological quality of both common and durum wheat

underlines the necessity of bug damage control before batch processing. The

practical tolerance for bug-damaged kernels in industry, regardless of the wheat type

(common or durum) or variety, is 2-3% (Canhilal et al., 2006). The damage caused

by sunn pests can be detected through a visual inspection of the kernels which are

characterized by a discoloured halo around the stylet penetration point (Critchley,

1998). Unfortunately, visual damage detection is often not completely reliable, since

it is strongly related to the operator’s experience and sensitivity. Moreover, similar

symptoms can be sometimes due to the activity of pathogens or the abnormal starch

deposition. As a consequence, visual inspection should be associated with complex

biochemical analyses which assess protein degradation due to insect attack, such as

reverse-phase or size-exclusion high performance liquid chromatography and free

zone capillary or gel electrophoresis (Sivri et al., 1999; Aja et al., 2004; Rosell et al.,

2002a; Vaccino et al., 2006).

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At the technological level, one of the most specific methods to detect bug damage on

grains and flour is the sodium dodecyl sulphate sedimentation volume test (SSV)

proposed by Every (1992).

According to the European ISO605 and the United States Department of Agriculture

(USDA) standards, the food supply chain requires practical and reliable screening

procedures in order to ensure the technological quality and marketability of wheat

kernels and flour. Mixolab® (Chòpin Technologies, Villeneuve la Garenne, France) is

a recent instrument that is used to determine the rheological quality of flour and to

more accurately describe its behaviour during bread making. This device provides, in

one single test, a complex analysis of the rheological properties of wheat flour dough,

considering dough behaviour during mixing, protein coagulation, heating-up

behaviour at enzyme activity intensification, and starch gelatinization and

retrogradation during the final cooling. The instrument has also proved useful to

analyse the quality of durum wheat (Moscaritolo et al., 2008). Kahraman and Köksel

(2013a) have recently suggested a new and specific Mixolab® analytical protocol to

estimate bug damage in flour.

The aims of this study were to make the direct field comparison of the bug feeding

effect on both common and durum wheat and to evaluate the potential of Mixolab® to

detect bug damage on the two crops considering different analysis protocols.

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2. Material and Methods

2.1. Agronomic information

Field trials were carried out in Carignano, Piedmont, NW Italy (44°53'8.69"N,

7°41'16.75"E, 232 m a.s.l.), in a medium-texture fertile soil, during the 2012-13 and

2013-14 growing seasons. Common and durum wheat were cultivated side by side in

the same field, according to the normal crop management programme applied to

wheat in the growing area.

The common wheat cultivars were Generale (Consorzio nazionale sementi,

Conselice, RA, Italy) in 2012-13 and Arrocco (Limagrain Italia S.p.A., Busseto, PR,

Italy) in 2013-14, which are classified, according to the Italian bread-making quality

grade (Foca et al., 2007), as superior bread making-quality wheat. The durum wheat

cultivar were Colombo (Apsovsementi S.p.A., Voghera, PV, Italy) in 2012-13 and

Saragolla (Produttori Sementi Bologna S.p.A., Argelato, BO, Italy) in 2013-14, which

are classified as high quality wheat. Planting was conducted in 12 cm wide rows at a

seeding rate of 450 seeds m-2 on the last decade of October. The previous crop was

maize for grain every growing season. The weed control was conducted with

isoproturon and diflufenican at wheat tillering (growth stage, GS 23; Zadoks et al.,

1974), while fungicide treatments were performed to avoid the development of foliar

and head fungal diseases at stem elongation (GS 35, a.i. azoxystrobin and

cyproconazole applied at 0.2 kg ha-1 and 0.08 kg ha-1, respectively) and at heading

(GS 58, a.i. prothioconazole applied at 0.250 kg ha-1). A total of 170 kg N ha-1 was

applied as a granular ammonium nitrate fertilizer, split 50 kg N ha-1 at wheat tillering

(GS 23), 80 kg N ha-1 at stem elongation (GS 32) and 40 kg N ha-1 at booting (GS

46).

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2.2. Collection and rearing of cereal bugs

During spring 2013 and 2014, large quantities of E. maura were collected in several

wheat fields in Piedmont, and transferred to laboratories. There, they were reared on

kernels and small wheat plants inside 3 L plastic boxes. The lid of these boxes

(265 mm × 175 mm) was cut in the middle and closed with a net. Mass rearing was

conducted in climatic chambers maintained at 25 ± 1°C, 70 ± 5% RH and a 16L:8D

photoperiod.

2.3. Exposure of wheat to bug feeding

Two parallel experiments were performed in order to evaluate the effects of bug

feeding on common and durum wheat quality. These experiments involved

comparing the rheological parameters of the flour derived from: i) grain samples

obtained in the presence or absence of E. maura in field conditions (first experiment);

ii) grain samples with different percentages of bug-damaged kernels (second

experiment).

The first experiment was conducted during two growing seasons using large net

cages (4×4×3.5m) which were placed at early milk stage (GS 73) for both crops, after

a careful check for the absence of bugs. Two treatments, that are the presence of E.

maura and control without insects, and three replications for each treatment and crop

were performed according to a completely randomized experimental design. In 2013,

48 individuals (i.e., 3 individuals m-2) were introduced into each cage and left until the

end of wheat ripening (July 2). In 2014, 45 individuals were introduced into each

cage and left until the end of wheat ripening (July 1). At this stage, in both years, all

the bugs were removed and recorded through an accurate inspection of the cage

contents.

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On July 22, 2013 and July 11, 2014, the grain was obtained by harvesting with a

Walter Wintersteiger cereal plot combine-harvester, and 2 kg grain samples were

taken from each cage for the qualitative analyses. Damaged kernels, i.e. the

percentage of seeds showing, on visual inspection, the typical discoloured area

around the point of bug stylet penetration, were determined on three 100-kernel

randomly selected samples for each treatment and replication.

In the second experiment, carried out in 2012-13 growing season, white sleeve

polythene fine mesh net cages were positioned in the field for both crops at early

maturity stage. In particular, 25 cages (0.5 m long, 0.2 m diameter) were suspended

from a pole in the ground, and the cage ends were closed over the vegetation to

prevent entry and escape of the cereal pest. On June 5, at early milk stage (GS 73),

a group of 20 spikes was included into each cage, and 1 adult, 5 3rd-instar juveniles

and 35 eggs were introduced, left for 27 days, and removed at the wheat ripening, in

order to obtain high amounts of bug-damaged kernels. The spikes from each cage

were hand harvested, placed in paper bags, and transferred to laboratories where

they were threshed with a Minibat Godé laboratory-sheller (Godé, Le Catelet,

France). The kernels were visually inspected to detect and keep only those showing

an evident dark puncture mark at the bug stylet penetration point, surrounded by a

discoloured area.

Five mixtures of sound kernels were obtained for each crop with an increasing

replacement rate (1%, 2.5%, 5%, 10%, 20%) of bug-damaged kernel, and, after an

accurate mixing, the kernels were employed for milling. Two replications of 600 g of

kernels were obtained for each replacement level. The undamaged kernels were

obtained from pre-defined field plots in which a pyrethroid insecticide (a.i.

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deltamethrin applied at 0.012 kg ha-1) had been applied at the beginning of the milk

stage (GS 71). These sound kernels were analyzed as a control.

2.4. Quality analyses

Grain moisture content was determined by means of a Dickey-John GAC2100 grain

analysis meter (Dickey-John Corp., Auburn, IL, USA) using the supplied programme.

Moisture calibration was checked using oven drying techniques.

Grain samples (300 g) from each plot were milled using a Retsch ZM 200 (Retsch

GmbH, Haan, Germany), fitted with a 1 mm aperture sieve, and the resulting

wholemeal was analysed by near-infrared reflectance spectroscopy, using a

NIRSystems 6500 monochromator (Foss-NIRSystems, Silver Spring, MD, USA). The

GPC (N x 5.7, dry matter basis) was determined according to AACC 39-10 (AACC,

2000).

Two kg and 0.6 kg grain samples were milled for each replication, for the first and

second experiments, respectively, with an experimental mill (Labormill 4RB, Bona,

Monza, Italy) after tempering according to the grain hardness. The applied milling

operation conditions, in particular the type of rolls and sieves, were specific for each

crop, common and durum wheat crop, according to the experimental mill protocol.

The rheological properties were studied on each flour sample using a Mixolab®

analyser (Chòpin Technologies, Villeneuve la Garenne, France), according to the

ICC Standard Method 173 (ICC, 2010). This instrument allows specific information to

be obtained on the behaviour of dough constituents (starch, protein, water) by

continuously measuring the torque (Nm) produced as the dough passes between two

mixing blades and is subject to the dual stress of mixing and temperature changes.

The test is based on the preparation of a constant dough sample to obtain a target

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consistency (1.1 Nm +/-0.05) during the first test phase. Samples were mixed

choosing the “Chopin+” protocol. The resulting Mixolab® curve is separated into five

different phases. A detailed description of the physical changes that occur during a

Mixolab® measurement (Chopin+ protocol) was reported in Rosell et al. (2007). The

key parameters derived from the Mixolab® curve are: water absorption capacity (WA,

%), which represents the amount of water that flour can absorb to obtain a pre-set

dough consistency to produce a torque (peak C1) of 1.1 Nm +/-0.05 at 30°C; Dough

Development Time (DDT, min), that is the time required to obtain the maximum

torque (C1) at 30°C; dough stability (min) or elapsed time, at which the produced

torque is kept at 1.1 Nm (± 11%); amplitude (Nm), which represents the width of

curve to C1 and refers to dough elasticity; C2 (Nm), which measures protein strength

after a decrease in dough consistency due to mechanical shear stress and

temperature increase (from 30°C to 60°C) and provides an indication of protein

quality; C3 (Nm), which measures starch gelatinization (starch granules absorb water

and the further increase in temperature, from 60°C to 90°C, results in an increase in

viscosity); C4 (Nm), which measures hot gel stability connected to the amylase

activity (at a constant temperature of 90°C); C5 (Nm), which measures starch

retrogradation in the cooling phase (from 90°C to 50°C); α, the slope of the curve

between the end of the period of 30°C and C2, and refers to the protein network

weakening rate (nm/min); β, the gelatinization rate (Nm/min) or the slope of the curve

between C2 and C3; γ, the cooking stability rate (Nm/min) or the slope of the curve

between C3 and C4.

In addition, flour samples were mixed applying the Mixolab “Wheatbug” protocol, as

proposed by Kahraman and Köksel (2013b). Briefly, the protocol is characterized by

a mixing period of 3 min at 110 rpm, incubation for 20 min without mixing and a

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mixing period of 5 min at 110 rpm at a constant temperature of 35°C. The decrease

in consistency between the 2.9th min and the 28th min (CMC: Change in Mixolab

Consistency) was calculated and expressed as a percentage.

A sodium dodecyl sulphate sedimentation volume test (SSV) was performed on the

same flour samples analysed with Mixolab®, according to Every (1992).

2.5. Statistical analyses

An analysis of variance (ANOVA) was used to compare the qualitative parameters

recorded in both experiments.

The residual normal distribution was verified using the Kolmogorov-Smirnov test,

while variance homogeneity was verified using the Levene test. In the second

experiment, multiple comparison tests were performed according to the Ryan-Einot-

Gabriel-Welsch F test on treatment means.

Simple correlation coefficients were obtained for all the Mixolab® parameters, relative

to each other and to SSV, while keeping the data sets together which referred to the

two experiments carried out in 2012-13 growing season, but considering common

and durum wheat separately. The relationship between the best Mixolab parameters

correlated to bug feeding damage and SSV was studied for each crop through linear

regression analysis.

The SPSS for Windows statistical package, Version 21.0 (SPSS Inc., Chicago, IL,

USA), was used for the statistical analyses.

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3. Results

In the first experiment, a direct comparison of qualitative damage to common and

durum wheat exposed to E. maura was made in field conditions. No significant

differences (ANOVA, P>0.05) in the number of bugs captured in the cages were

found between the two crops in 2012-2013 (76.67±27.67 for common wheat and

62.00±19.86 for durum wheat) and in 2013-2014 (145.67±15.38 for common wheat

and 115.33±18.89 for durum wheat).

The results of artificial infestation with E. maura, which was conducted for direct

comparison with an uninfested control in field conditions for 2012-13 and 2013-14

growing seasons on damaged kernel rate, GPC and the Mixolab parameters are

reported in Table 1.

In 2012-13 growing season, ANOVA showed a significant effect of the insect

infestation on the damaged kernel percentage, SSV, dough stability, C2, C3, C4 and

C5 points, α and β slopes, and CMC for both crops. The E. maura infestation also

significantly affected the WA and γ slope, but only for the durum wheat. The GPC

was not significantly different between the compared treatments for either crops.

On visual inspection, the average percentage of damaged kernels, after the artificial

infestation of E. maura, was 21.6% and 16.0% for common and durum wheat,

respectively. Compared to the control, which was characterized by less than 1% of

kernels with visual symptoms, insect feeding reduced SSV by 40% and 68%, for

common and durum wheat, respectively. As far as the Mixolab® parameters of

Chopin+ protocol are concerned, the bug damage led to a greater reduction in dough

stability for common (-65%) than durum wheat (-32%). On the other hand, the

parameters that describe the protein strength (the C2 point and α slope) were

affected more by insect activity in the durum wheat. The observed reductions in the

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C2 value were 40% and 56%, while those of the α value were 39% and 44%, for

common and durum wheat, respectively. C3, C4 and C5, which describe starch

behaviour to temperature modification, also resulted in a greater decrease, as a

result of the insect presence, in durum than in common wheat. When considering the

Wheatbug Mixolab® protocol, the insect feeding activity increased the CMC by 34%

and 24% for common and durum wheat, respectively.

In 2013-14 growing season, ANOVA confirmed a significant effect of the insect

infestation on the damaged kernel percentage, SSV, dough stability, C2 and CMC for

both crops. Although the average percentage of damaged kernels, after the artificial

infestation of E. maura, was higher compared to that observed in the previous

growing season, the rheological impact measured through SSV or Mixolab® test was

generally lower. Compared to the control, insect feeding reduced SSV by 5% and

66%, dough stability by 12% and 30%, and C2 value by 12% and 16%, for common

and durum wheat, respectively. Moreover, the insect feeding activity increased the

CMC by 12% and 11% for common and durum wheat, respectively.

In the second experiment, the increasing percentages of damaged kernels (i.e., with

visual damage of bug feeding) led to a significant decrease in SSV, dough stability,

the C2 and C3 values, and CMC for both common and durum wheat (Table 2). For

both crops, the effect of a reduction in protein quality, measured through the SSV

test, began at 1% of replacement with kernels damaged by insect feeding. The SSV

values at 1% of damaged kernels were 7% and 11% lower than the control (i.e.,

sound samples with a 0% replacement level), for common and durum wheat,

respectively. The durum showed a greater reduction in SSV at each replacement

level compared to common wheat, thus underlining a probable higher susceptibility of

this crop to the E. maura feeding activity on grains.

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As far as the Mixolab® Chopin+ protocol is concerned, dough stability and the C2

value were confirmed to be the most closely related parameters to bug damage for

both crops, although they showed a lower capacity to stress the insect damage than

SSV. A significant reduction in dough stability and protein strength (C2) was

observed for common wheat at the 2.5% and 5% replacement levels, respectively.

On the other hand, the C2 value was significantly lower for durum wheat than the

control at 2.5%, while only at 5% of substitution did the dough stability significantly

decrease. At 5% of kernel substitution, the dough stability was reduced by 33% and

25% for common and durum wheat, respectively. The reduction in protein strength

(C2) was 12% and 28% for common and durum wheat, respectively at the same rate.

Starch gelatinisation (C3) was affected by the percentage of bug-damaged kernels at

the 5% level for common wheat, while it was only influenced significantly at the 20%

level for durum wheat.

Conversely, C4, C5 and β varied significantly with the increase in damaged kernels

for common wheat, but the differences in these parameters were only more

pronounced for the highest replacement levels. Only for durum wheat, but at 20% of

substitution, did the protein network wakening rate (α) show a significant difference,

compared to the control. The WA, DDT, amplitude and cooking stability rate (γ) were

unaffected by the percentage of damaged kernels.

The CMC parameter obtained using the Wheatbug protocol was confirmed to be well

able to describe bug damage and showed similar results to those observed with the

Chopin+ protocol for dough stability and the C2 value: the analysis showed a

significantly higher CMC at 2.5% of substitution compared to the control for both

crops.

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Table 3 reports the correlation coefficients and their significances for the Mixolab®

parameters and SSV for flour samples derived from both the first and the second

experiment carried out on cvs Generale and Colombo in 2012-13 growing season.

SSV resulted to be significantly correlated to dough stability, the protein strength

(C2), the protein network wakening rate (α) and CMC for both common and durum

wheat. The correlation was always highly significant (P<0.01), with the exception of α

for common wheat (P<0.05). Moreover, a significant correlation was observed

between SSV and C3, C4, C5, β and γ, but only for common wheat. As expected, the

different Mixolab® parameters were often significantly correlated to each other.

The relationship between SSV and dough stability, the C2 value, the α value and

CMC is shown in Figure 1 for both considered crops. The regression analysis for the

reported equations was always significant. The highest R2 values were observed for

the relationship between SSV and protein strength (C2) for both common (R2 = 0.87,

P<0.01) and durum (R2 = 0.93, P<0.001) wheat.

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4. Discussion

The present study underlines that Mixolab® can properly estimate and describe

damage caused by sunn pests in both common and durum wheat. The occurrence of

enzymatic degradation of the gluten proteins, caused by E. maura, clearly affected

the parameters that are closely related to gluten content and composition: dough

stability, protein strength (C2), protein network wakening rate (α). These changes in

dough rheological properties are related to a loss in gluten functionality, resulting

from complex changes in the gluten structure (Pérez et al., 2005; Torbica et al.,

2014). The previously reported Mixolab® parameters are closely correlated to SSV

and were clearly able to describe the occurrence of bug damage in each wheat

species. The Mixolab® parameter most closely related to bug damage, expressed as

SSV, for both considered crops was protein strength.

Moreover, a clear effect of insect infestation was also observed on starch

gelatinization (C3), hot gel stability (C4), starch retrogradation in the cooling phase

(C5) and starch gelatinisation rate (β) for both crops, particularly in the first

experiment for the 2012-13 growing season. Conversely, in the second experiment,

considering the different replacement levels, the impact of the increasing percentage

of bug-damaged kernels on the previously reported starch parameters was more

evident on common wheat than on durum wheat. The effects of the alteration of

starch granules have already been reported for Nysius spp. (Heteroptera:

Lygaeidae), a bug species noxious to wheat, in Australia and New Zealand (Every et

al., 1990; Lorenz and Meredith, 1988), although no differences in diastatic activity

were assessed. However, amylase activity has not been reported to be involved in

wheat damage caused by Aelia spp. or Eurygaster spp., and starch granules

examined by means of scanning electron microscopy were also intact in the

19

surrounding areas of the insect puncture (Rosell et al., 2002b). Thus, it can be

supposed that the observed different dough behaviour, concerning the starch quality

Mixolab® parameters, is a consequence of gluten alteration due to E. maura in the

endosperm protein-starch matrix rather than a direct effect on starch granules.

Although the quality of gluten, as well as the quantity, has a clear impact on dough

water absorption and development time (Cauvain and Young, 2000), the occurrence

of E. maura damage, obtained through an artificial infestation or by increasing

substitution of damaged kernels, did not here result in an impact on WA or DDT, with

the exception of the WA of durum wheat in the first experiment in the 2012-13

growing season. This result is in contrast with the results of Hariri et al. (2000), who

reported a proportional increase in WA and decrease in DDT for a rise in the

damaged kernel percentage.

As far as the specific Mixolab® Wheatbug protocol is concerned, the CMC parameter

seems to provide clear evidence of bug damage, especially for common wheat,

which results in a higher correlation to dough stability and protein strength (C2) than

SSV. Conversely, for durum wheat, the relationship between the Mixolab® rheological

parameters and SSV is closer than the relationship between these parameters and

CMC.

A close positive relationship between bug density and the percentage of kernel

damage in common and durum wheat was reported in previous studies on E.

integriceps Puton (Heteroptera: Scutelleridae), along with a negative relationship

between increasing bug-damaged kernels and flour quality parameters (Canhilal et

al., 2006; Karababa and Ozan, 1998). The present research has shown that the

detrimental effects of bug damage on the rheological quality of durum wheat are

quite similar to those that occur on common wheat. These data, obtained by means

20

of the Mixolab® analysis, confirm the results of Olanca and collaborators (2009) who

considered the impact of sunn pests on protein composition and on the structure of

durum wheat. However, the effect of E. maura on the SSV parameter was generally

higher in durum wheat than in common wheat, considering both growing seasons

and different cultivars. Comparing different common wheat varieties, Kinaci and

Kinaci (2004) demonstrated a variation in quality damage due to sunn pests among

wheat varieties. The authors reported that, in terms of sedimentation values, hard

grains suffered more than soft ones after bug infestations.

Mixolab® pointed out significant differences on the parameters of dough stability,

protein strength and CMC in both growing seasons and for both crops, even

considering different cultivars. However, in the 2013-14 growing season the

rheological impact, measured through SSV or Mixolab® dough stability, protein

strength and CMC test, was lower even if the average percentage of damaged

kernels was higher compared to that observed in the previous growing season.

This effect should not be related only to the use of different cultivars, which have

similar level of quality. A possible explanation could be related to the meteorological

conditions that occurred during wheat ripening: the 2013-14 growing season was

characterized by frequent rainfall and lower temperatures from flowering to

harvesting compared to the 2012-13 season. These conditions may have contributed

to change the quality of kernel proteins or led to a higher fungal kernel infection, with

an overestimation of visual insect damage. Nevertheless, both SSV and Mixolab®

evaluations rely upon the content and quality of kernel proteins, which is related to

the environmental and agronomic conditions; as a consequence, such methods only

indirectly and non-specifically define gluten damage caused by bugs. A specific

quantification of damage caused by bugs should be addressed with biochemical

21

methods able to assess protein degradation due to insect attack. An immunological

method for a quick and direct detection of bug contamination on flour batches, as

preliminarily reported (Vaccino et al., 2008), could be a specific and effective tool to

detect bug damage.

As far as the tolerance of bug-damaged kernel is concerned, this study has also

confirmed for durum wheat results obtained in other studies referring to the effect of

bug-damaged kernel percentage to dough quality parameters of common wheat.

Karababa and Ozan (1998) and Hariri et al. (2000), in experiments carried out in

Turkey and Syria, respectively, reported the physicochemical properties (farinograph

stability time, mixograph development time) of common wheat samples with more

than 5% bug-damaged kernels changed remarkably and showed significantly lower

bread-making quality. In the present experiments, the percentage of bug-damaged

kernels clearly affected the quality of both common and durum wheat, although the

main rheological parameters (dough stability for common wheat, C2 for durum

wheat) and CMC obtained with the Mixolab® Wheatbug protocol were already

noticeable at the 2.5% addition of damaged kernel level. These results confirm the

study of El-Haramain et al. (1984), who reported that a 2-3% grain damage clearly

reduces the acceptability of flour for baking purposes. It can therefore be seen that

the SSV analysis has been able to stress a significant difference, compared to the

control even at a 1% replacement level with damaged kernels, thus confirming the

remarkable ability of this test to detect the bug damage of flour.

Ozderen et al. (2008) reported, for durum wheat, that the pasta processing industry

could tolerate a relatively higher level of bug damage than the bread processing

industry. Furthermore, the authors compared quite high bug damage levels (around

20% and 40%) with a sound control (0%), with reference to spaghetti cooking quality.

22

The collected data suggest that for both wheat species (common and durum) the

presence of 2.5-5% of damaged kernels is sufficient to seriously affect their quality.

Petrova (2002) reported that, depending on the variety, bug damage negatively

affected the cooking quality of durum wheat: no significant changes were observed

for high gluten quality cultivars, even at a 5% damage level, while a 2% damage level

affected pasta quality in weak gluten cultivars.

In conclusion, Mixolab® can be considered a suitable tool for a quick detection of

damage caused by sunn pests in common and durum wheat, as it shows a clear

correlation to the percentage of damaged kernels. The reduction in rheological

quality evaluated through this device was similar for both crops, although the

detrimental effects of bug damage on the SSV parameter were greater in durum

wheat than in common wheat in both growing seasons.

23

Acknowledgements

The authors would like to thank Alessandra Gazzola, Matteo Calcagno and Valentina

Sovrani for their precious help and cooperation in the field and laboratory work.

The research was conducted with the financial support of the Regione Piemonte, as

part of the QUALICHAIN project.

24

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28

Tables

Table. 1

Effect of cereal bug infestation on damaged kernel, grain protein content (GPC), sodium dodecyl sulphate sedimentation volume

(SSV) and Mixolab parameters of flour and semolina derived from common and durum wheat, respectively, in 2012-13 and 2013-

14 growing seasons.

Year Crop E. maura

Damaged

Chopin+ protocola

Wheatbug protocolb

infestation kernels GPC SSV WA DDT Amplitude Stability C2 C3 C4 C5 α β γ CMC (%) (%) (ml) (%) (min) (Nm) (min) (Nm) (Nm) (Nm) (Nm) (Nm/min) (Nm/min) (Nm/min) (%)

2013 Common Control 0.8 14.0 58 56.9 1.8 0.08 10.0 0.54 2.09 1.90 3.11 -0.09 0.69 -0.04 18

wheat Artificial infestation 21.6 13.9 38 56.8 1.7 0.08 3.5 0.32 1.76 1.66 2.65 -0.05 0.17 -0.04 52

P (F) < 0.001 0.896 < 0.001 0.141 0.212 0.667 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.935 < 0.001

semc 2.0 0.4 1.7 0.1 0.2 0.01 0.428 0.019 0.03 0.04 0.09 0.03 0.08 0.03 1.8

Durum Control 0.6 13.4 36 58.8 1.3 0.11 3.2 0.28 1.25 0.72 1.17 -0.07 0.32 -0.07 41

wheat Artificial infestation 16.0 13.8 13 63.0 1.2 0.10 2.2 0.12 0.92 0.55 0.86 -0.04 0.24 -0.05 65

P (F) < 0.001 0.102 < 0.001 < 0.001 0.137 0.291 0.018 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.007 0.046 < 0.001

semc 2.2 0.2 1.7 0.8 0.1 0.01 0.7 0.01 0.03 0.04 0.09 0.02 0.04 0.01 1.4

2014 Common Control 1.67 14.5 87.7 57.8 2.47 0.07 10.33 0.58 2.23 1.70 3.36 -0.06 0.41 -0.29 12 wheat Artificial infestation 27.9 14.3 82.9 57.8 2.35 0.08 9.07 0.51 2.09 1.62 3.46 -0.04 0.40 -0.14 23

P (F) < 0.001 0.346 < 0.001 0.778 0.590 0.101 0.019 < 0.001 < 0.001 0.126 0.449 0.640 0.925 0.374 < 0.001 semc 4.6 0.3 0.70 0.31 0.31 0.01 0.47 0.02 0.01 0.06 0.16 0.03 0.20 0.21 1.8

Durum Control 2.4 13.9 65.3 64.8 1.41 0.11 3.96 0.25 0.96 0.25 0.43 -0.06 0.22 -0.06 44 wheat Artificial infestation 35.9 13.9 22.3 64.2 1.29 0.10 2.76 0.21 0.95 0.17 0.30 -0.03 0.21 -0.05 54

P (F) < 0.001 0.877 < 0.001 0.207 0.220 0.519 0.021 0.028 0.704 0.200 0.307 0.102 0.502 0.686 0.027 semc 2.3 0.3 3.13 0.60 0.11 0.01 0.46 0.02 0.03 0.05 0.11 0.01 0.02 0.01 4.4

29

Reported values are based on 3 replications. a Mixolab parameters Chopin+ protocol: WA = water absorption, the amount of water required in dough development; DDT =

Dough Development Time; stability = time of dough stability at constant temperature; amplitude = dough elasticity; C2 = protein

strength; C3 = starch gelatinisation; C4 = hot gel stability; C5 = starch retrogradation in the cooling phase; α = protein network

weakening rate ; β = starch gelatinisation rate; γ = cooking stability rate. C2, C3, C4 and C5: end points of the corresponding

mixing stages. b Mixolab parameter “Wheatbug” protocol: CMC = Change in Mixolab Consistency c sem = standard error of mean.

30

Table. 2

Effect of the replacement level of common and durum wheat kernels with cereal bug damaged ones on sodium dodecyl sulphate

sedimentation volume (SSV) and the Mixolab parameter using the Chopin+ and Wheatbug protocol.

Chopin+ protocola Wheatbug protocolb

Crop Damaged kernel SSV WA DDT Amplitude Stability C2 C3 C4 C5 α β γ CMC

replacement level (mL) (%) (min) (Nm) (min) (Nm) (Nm) (Nm) (Nm) (Nm/min) (Nm/min) (Nm/min) (%)

Common 0 50 A 57.6 a 1.5 a 0.10 a 8.2 a 0.47 a 2.00 a 1.83 a 2.93 a -0.08 a 0.55 a -0.05 a 32 d

wheat 1 47 B 57.9 a 1.7 a 0.09 a 8.0 a 0.48 a 1.99 a 1.79 ab 2.86 ab -0.07 a 0.56 a -0.04 a 34 d

2.5 46 bc 57.6 a 1.5 a 0.10 a 6.6 b 0.45 ab 1.96 a 1.80 ab 2.87 ab -0.08 a 0.57 a -0.06 a 37 c

5 44 c 57.3 a 1.4 a 0.09 a 5.5 c 0.42 b 1.89 b 1.72 bc 2.73 ab -0.07 a 0.53 a 0.00 a 44 b

10 41 d 57.6 a 1.9 a 0.08 a 4.1 d 0.37 c 1.82 c 1.71 bc 2.68 ab -0.05 a 0.50 a -0.01 a 49 ab

20 32 e 57.8 a 1.9 a 0.08 a 2.5 e 0.28 d 1.71 d 1.64 d 2.54 b -0.05 a 0.05 b -0.01 a 54 a

P (F) <0.001 0.417 0.088 0.199 <0.001 <0.001 <0.001 0.004 0.032 0.120 0.004 0.063 <0.001

semc 1.3 0.44 0.31 0.02 0.22 0.02 0.03 0.05 0.16 0.02 0.14 0.03 3.0

Durum 0 27 a 58.1 a 1.0 a 0.09 a 2.5 a 0.25 a 1.34 a 0.75 a 1.19 a -0.05 a 0.46 a -0.07 a 44 c

wheat 1 24 b 58.1 a 1.0 a 0.08 a 2.3 a 0.24 ab 1.33 a 0.74 a 1.15 a -0.05 a 0.40 a -0.06 a 47 c

2.5 20 c 57.9 a 1.0 a 0.09 a 2.1 ab 0.20 bc 1.31 ab 0.74 a 1.15 a -0.05 ab 0.43 a -0.08 a 52 b

5 17 d 58.2 a 1.1 a 0.07 a 1.8 b 0.18 c 1.30 ab 0.77 a 1.17 a -0.04 ab 0.43 a -0.06 a 54 ab

10 13 e 57.5 a 1.0 a 0.08 a 1.7 b 0.16 c 1.29 ab 0.77 a 1.21 a -0.04 ab 0.43 a -0.07 a 56 ab

20 11 e 58.7 a 1.2 a 0.08 a 1.1 c 0.12 d 1.19 b 0.74 a 1.17 a -0.03 b 0.43 a -0.05 a 59 a

P (F) <0.001 0.057 0.608 0.316 <0.001 <0.001 0.048 0.602 0.739 0.050 0.927 0.098 <0.001

semc 1.0 0.63 0.27 0.02 0.29 0.03 0.07 0.07 0.09 0.01 0.11 0.02 2.5

Means followed by different letters are significantly different (the level of significance is shown in the table). Reported values are

based on 2 replications. The experiment was carried out in the 2012-13 growing season. a Mixolab parameters Chopin+ protocol: WA = water absorption, the amount of water required in dough development; DDT =

Dough Development Time; stability = time of dough stability at constant temperature; amplitude = dough elasticity; C2 = protein

strength; C3 = starch gelatinisation; C4 = hot gel stability; C5 = starch retrogradation in the cooling phase; α = protein network

31

weakening rate ; β = starch gelatinisation rate; γ = cooking stability rate. C2, C3, C4 and C5: end points of the corresponding

mixing stages. b Mixolab parameter “Wheatbug” protocol: CMC = Change in Mixolab Consistency c sem = standard error of mean.

32

Table. 3

Correlation matrix between the sodium dodecyl sulphate sedimentation volume (SSV) and the Mixolab parametersa, calculated

separately for common and durum wheat.

Parameters WA DDT Amplitude Stability C2 C3 C4 C5 α β γ CMC SSV

WA 0.056 0.198 -0.090 -0.082 -0.092 -0.116 -0.230 0.164 -0.026 0.088 0.186 -0.360

DDT 0.497 -0.852** -0.295 -0.329 -0.333 -0.265 -0.261 0.457 -0.358 0.347 0.196 -0.183

Amplitude 0.447 0.478 0.573 0.547 0.575 0.536 0.499 -0.669 0.448 -0.588 -0.462 0.314

Stability 0.095 0.199 0.785* 0.985** 0.993** 0.981** 0.974** -0.925** 0.854** -0.549 -0.975** 0.910**

C2 -0.412 -0.089 0.442 0.851** 0.996** 0.970** 0.962** -0.906** 0.929** -0.497 -0.954** 0.935**

C3 -0.964** -0.566 -0.369 0.089 0.583 0.983** 0.976** -0.931** 0.899** -0.552 -0.968** 0.929**

C4 -0.979** -0.451 -0.544 -0.166 0.341 0.938** 0.993** -0.936** 0.857** -0.595 -0.984** 0.931**

C5 -0.971** -0.307 -0.394 -0.060 0.432 0.921** 0.974** -0.936** 0.842** -0.605 -0.985** 0.956**

α 0.166 -0.272 -0.654 -0.930** -0.910** -0.297 -0.103 -0.237 -0.753* 0.606 0.938** -0.830*

β -0.874** -0.632 -0.668 -0.403 0.111 0.836** 0.893** 0.828* 0.229 -0.345 -0.812* 0.892**

γ 0.545 0.527 -0.209 -0.422 -0.569 -0.602 -0.470 -0.452 0.428 -0.343 0.506 -0.471

CMC 0.672 0.130 -0.189 -0.615 -0.929** -0.783** -0.618 -0.713* 0.784* -0.404 0.517 -0.928**

SSV -0.241 0.157 0.594 0.909** 0.966** 0.402 0.175 0.301 -0.962** -0.073 -0.422 -0.870**

(*) = correlation significant at P ≤ 0.05; (**) correlation significant at P ≤ 0.01. Data reported in table are Pearson product-

moment correlation coefficient.

The data with a gray background refer to durum wheat, while those with a white background refer to common wheat. The

reported data for each crop refer to samples obtained from both the first and the second experiment in the 2012-13 growing

season.

33

a Mixolab parameters Chopin+ protocol: WA = water absorption, the amount of water required in dough development; DDT =

Dough Development Time; stability = time of dough stability at constant temperature; amplitude = dough elasticity; C2 = protein

strenght; C3 = starch gelatinisation; C4 = hot gel stability; C5 = starch retrogradation in the cooling phase; α = protein network

wakening rate ; β = starch gelatinisation rate; γ = cooking stability rate. C2, C3, C4 and C5: end points of the corresponding

mixing stages. Mixolab parameter “Wheatbug” protocol: CMC = Change in Mixolab Consistency.

Figure

Figure. 1.

Relationship between sodium dodecyl sulphate sedimentation volume (SSV) and Mixolab

stability, protein weakness (C2 point), protein network weakening rate (α slope) and

Change in Mixolab Consistency (CMC) for common and durum wheat.

(*) = regression significant at P ≤ 0.05; (**) regression significant at P ≤ 0.01; (**)

regression significant at P ≤ 0.001. R2 = coefficient of determination of regression analysis.

The reported data for each crop refer to samples obtained from both the first and the

second experiment in the 2012-13 growing season.